Biophysical Mimicry of Lung Surfactant Protein B by Random Nylon-3

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Biophysical Mimicry of Lung Surfactant Protein B by Random Nylon-3 Copolymers Michelle T. Dohm,† Brendan P. Mowery,‡ Ann M. Czyzewski,§ Shannon S. Stahl,*,‡ Samuel H. Gellman,*,‡ and Annelise E. Barron*,§,| Department of Chemistry, Northwestern UniVersity, 2145 North Sheridan Road, EVanston, Illinois 60208-3100, Department of Chemistry, UniVersity of WisconsinsMadison, 1101 UniVersity AVenue, Madison, Wisconsin, 53706, Department of Chemical and Biological Engineering, Northwestern UniVersity, 2145 North Sheridan Road, EVanston, Illinois 60208-3100, and Department of Bioengineering, Stanford UniVersity, W300B James H. Clark Center, 318 Campus DriVe, Stanford, California 94305-5440 Received November 22, 2009; E-mail: [email protected]; [email protected]; [email protected]

Abstract: Non-natural oligomers have recently shown promise as functional analogues of lung surfactant proteins B and C (SP-B and SP-C), two helical and amphiphilic proteins that are critical for normal respiration. The generation of non-natural mimics of SP-B and SP-C has previously been restricted to step-by-step, sequence-specific synthesis, which results in discrete oligomers that are intended to manifest specific structural attributes. Here we present an alternative approach to SP-B mimicry that is based on sequencerandom copolymers containing cationic and lipophilic subunits. These materials, members of the nylon-3 family, are prepared by ring-opening polymerization of β-lactams. The best of the nylon-3 polymers display promising in vitro surfactant activities in a mixed lipid film. Pulsating bubble surfactometry data indicate that films containing the most surface-active polymers attain adsorptive and dynamic-cycling properties that surpass those of discrete peptides intended to mimic SP-B. Attachment of an N-terminal octadecanoyl unit to the nylon-3 copolymers, inspired by the post-translational modifications found in SP-C, affords further improvements by reducing the percent surface area compression to reach low minimum surface tension. Cytotoxic effects of the copolymers are diminished relative to that of an SP-B-derived peptide and a peptoidbased mimic. The current study provides evidence that sequence-random copolymers can mimic the in vitro surface-active behavior of lung surfactant proteins in a mixed lipid film. These findings raise the possibility that random copolymers might be useful for developing a lung surfactant replacement, which is an attractive prospect given that such polymers are easier to prepare than are sequence-specific oligomers.

Introduction

Lung surfactant proteins SP-B and SP-C are required for the biophysical activity of lung surfactant (LS), the complex lipid-protein mixture that coats the internal air-liquid (a/l) interface of the vertebrate lung and reduces the work of breathing.1,2 Both proteins (∼1.5-2.0 combined weight percent of natural LS) contain a high proportion of lipophilic residues, and they adopt amphiphilic and helical conformations. The main function of LS is to regulate surface tension (γ, mN m-1) in the alveoli, tiny sacs that mediate gas exchange between the blood and air spaces of the lung, by optimizing available surface area, maximizing lung compliance, and stabilizing the alveolar

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Department of Chemistry, Northwestern University. University of WisconsinsMadison. § Department of Chemical and Biological Engineering, Northwestern University. | Stanford University. (1) Creuwels, L.; vanGolde, L. M. G.; Haagsman, H. P. Lung 1997, 175, 1–39. (2) Notter, R. H. Lung Surfactants: Basic Science and Clinical Applications; Marcel Dekker: New York, 2000. ‡

10.1021/ja909734n  2010 American Chemical Society

network against collapse.3 Three crucial functional characteristics of LS are as follows: (1) rapid adsorption to the a/l interface, (2) near-zero γ upon film compression, and (3) efficient respreading of material and minimal loss to the subphase through multiple breathing cycles.4 Deficient or dysfunctional LS results in infant or acute respiratory distress syndromes (IRDS or ARDS, respectively).5,6 Although no current exogenous treatment for ARDS exists,7 IRDS can be successfully treated with porcine- or bovinederived surfactant replacement therapies (SRTs).8 Use of animalderived substances, however, is nonideal because of the risk of zoonotic infection and the high cost of large-scale extraction, (3) Orgeig, S.; Bernhard, W.; Biswas, S. C.; Daniels, C. B.; Hall, S. B.; Hetz, S. K.; Lang, C. J.; Maina, J. N.; Panda, A. K.; Perez-Gil, J.; Possmayer, F.; Veldhuizen, R. A.; Yan, W. Integr. Comp. Biol. 2007, 47, 610–627. (4) Mingarro, I.; Lukovic, D.; Vilar, M.; Perez-Gil, J. Curr. Med. Chem. 2008, 15, 393–403. (5) Avery, M. E.; Mead, J. Am. J. Dis. Child. 1959, 97, 517–523. (6) Pison, U.; Seeger, W.; Buchhorn, R.; Joka, T.; Brand, M.; Obertacke, U.; Neuhof, H.; Schmit-Nauerburg, K. P. Am. ReV. Respir. Dis. 1989, 140, 1033–1039. (7) Lewis, J. E.; Jobe, A. H. Am. ReV. Respir. Dis. 1993, 147, 218–233. (8) Moya, F. R.; Maturana, A. Clin. Perinatol. 2007, 34, 145–177. J. AM. CHEM. SOC. 2010, 132, 7957–7967

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isolation, and purification. To eliminate dependence on animalderived material, many groups have sought to develop biomimetic LS replacements based on synthetic surfactant protein analogues.4 This approach could lead to a safe and bioavailable alternative to SRTs that may be able to treat or mitigate both IRDS and ARDS. SP-B in monomeric form is a 79-residue protein (8.7 kDa) with a net cationic charge and is postulated to contain four or five facially amphiphilic helices. SP-B forms four disulfide bonds: three intramolecular connections that presumably constrain conformational flexibility and one intermolecular bond that results in homodimerization.9-12 SP-C contains just 35 residues and forms a single helix.13 This protein has two palmitoylation points (positions 5 and 6), two cationic residues (11 and 12), and an extremely lipophilic polyvaline helix that approximates the length necessary for spanning a lipid bilayer.12,14-17 The sequences and structural attributes of both proteins are highly conserved across mammalian species, implying that these features are necessary for their ability to organize and regulate lipid film formation and to anchor the film to the a/l interface.3,18 Unfortunately, these attributes render the proteins very troublesome to obtain on a large scale by extraction or chemical synthesis; efforts to synthesize SP-B or SP-C or fragments thereof are often hampered by misfolding or aggregation.4 The main approaches toward functional mimicry of surfactant proteins have involved peptide fragment synthesis, limited dimerization of SP-B and its analogues, recombinant protein expression, and more recently, peptoid synthesis, the subject of our recent contributions in this field.19-24 Although a recombinant form of SP-C is available,25 it is not palmitoylated, and no recombinant form of SP-B has yet been reported. Chemically synthesized, surface-active peptide fragments of SP-B such as SP-B1-2526,27 and the dimeric constructs dSP(9) Hawgood, S.; Schiffer, K. Annu. ReV. Physiol. 1991, 53, 375–394. (10) Vandenbussche, G.; Clercx, A.; Clercx, M.; Curstedt, T.; Johansson, J.; Jornvall, H.; Ruysschaert, J. M. Biochemistry 1992, 31, 9169–9176. (11) Beck, D. C.; Ikegami, M.; Na, C. L.; Zaltash, S.; Johansson, J.; Whitsett, J. A.; Weaver, T. E. J. Biol. Chem. 2000, 275, 3365–3370. (12) Haagsman, H. P.; Diemel, R. V. Comp. Biochem. Physiol., Part A: Mol. Integr. Physiol. 2001, 129, 91–108. (13) Johansson, J.; Szyperski, T.; Curstedt, T.; Wuthrich, K. Biochemistry 1994, 33. (14) Johansson, J.; Curstedt, T.; Robertson, B. Eur. Respir. J. 1994, 7, 372– 391. (15) Creuwels, L. A.; Boer, E. H.; Demel, R. A.; van Golde, L. M. G.; Haagsman, H. P. J. Biol. Chem. 1995, 270, 16225–16229. (16) Kramer, A.; Wintergalen, A.; Sieber, M.; Galla, H. J.; Amrein, M.; Guckenberger, R. Biophys. J. 2000, 78, 458–465. (17) Bi, X. H.; Flach, C. R.; Perez-Gil, J.; Plasencia, I.; Andreu, D.; Oliveira, E.; Mendelsohn, R. Biochemistry 2002, 41, 8385–8395. (18) Perez-Gil, J. Biochim. Biophys. Acta 2008, 1778, 1676–1695. (19) Wu, C. W.; Seurynck, S. L.; Lee, K. Y. C.; Barron, A. E. Chem. Biol. 2003, 10, 1057–1063. (20) Seurynck, S. L.; Patch, J. A.; Barron, A. E. Chem. Biol. 2005, 12, 77–88. (21) Seurynck-Servoss, S. L.; Dohm, M. T.; Barron, A. E. Biochemistry 2006, 45, 11809–11818. (22) Seurynck-Servoss, S. L.; Brown, N. J.; Dohm, M. T.; Wu, C. W.; Barron, A. E. Coll. Surf. B. Biointerfaces 2007, 57, 37–55. (23) Brown, N. J.; Wu, C. W.; Seurynck-Servoss, S. L.; Barron, A. E. Biochemistry 2008, 47, 1808–1818. (24) Dohm, M. T.; Seurynck-Servoss, S. L.; Seo, J.; Zuckermann, R. N.; Barron, A. E. Biopolymers (Peptide Sci.) 2009, 92, 538–553. (25) Hawgood, S.; Ogawa, A.; Yukitake, K.; Schlueter, M.; Brown, C.; White, T.; Buckley, D.; Lesikar, D.; Benson, B. J. Am. J. Respir. Crit. Care Med. 1996, 154, 484–490. (26) Waring, A.; Taeusch, H. W.; Bruni, R.; Amirkhanian, J. D.; Fan, B. R.; Stevens, R.; Young, J. Pept. Res. 1989, 2, 308–313. 7958

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B1-2528 and “Mini-B”29 have demonstrated in vitro and in vivo success, but the challenge of generating these materials on a large scale is a stumbling block to pharmaceutical development. The chronic problem of achieving the desired extent of dimerization and multiple amphiphilic helices when mimicking SP-B has prompted recent endeavors to determine whether the incorporation of dimerization points can be circumvented while retaining good surfactant activity.24,30 The high cost of large-scale, stepwise synthesis and purification represents a significant barrier to the development of peptide-based drugs and has generated an interest in alternatives for use in an LS replacement.4 Non-natural oligomers, such as peptoids,31 β-peptides,32,33 and R/β-peptides34,35 can circumvent some peptide-associated problems, including irreversible aggregation and protease susceptibility; however, stepwise synthesis is required for preparation of these sequence-specific oligomers, and reversed-phase high-performance liquid chromatography (RP-HPLC) is necessary for their purification.32,36 Therefore, although these types of peptide mimics can display impressive biological activities and thereby shed light on relationships between molecular structure and resultant biophysical activities, sequence-specific non-natural oligomers do not alleviate the production cost problem. It is generally assumed that the function of a protein depends upon the sequence of amino acid residues and the threedimensional arrangement of amino acid side chains (or a subset thereof) that results from adoption of a specific secondary or tertiary structure. Recently, however, we proposed that discrete sequences and folding patterns may not be necessary for mimicry of the cell type-selective toxicity of host-defense peptides.37,38 Many of these natural antimicrobial peptides adopt helical conformations upon interaction with bacterial membranes and ultimately compromise the barrier function of the membrane; these peptides are generally selective as membranedisrupting agents, acting on bacteria in preference to eukaryotic cells.39 We have shown that nylon-3 copolymers (poly-βpeptides) containing sequence-random mixtures of lipophilic and cationic subunits can mimic the selective antibacterial activity (27) Bruni, R.; Taeusch, H. W.; Waring, A. J. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 7451–7455. (28) Veldhuizen, E. J. A.; Waring, A. J.; Walther, F. J.; Batenburg, J. J.; van Golde, L. M. G.; Haagsman, H. P. Biophys. J. 2000, 79, 377– 384. (29) Waring, A. J.; Walther, F.; Gordon, L. M.; Hernandez-Juviel, J.; Hong, T.; Sherman, M. A.; Alonso, C.; Alig, T.; Brauner, J. W.; Bacon, D.; Zasadzinski, J. J. Pept. Res. 2005, 66, 364–374. (30) Dohm, M. T.; Brown, N. J.; Seurynck-Servoss, S. L.; Bernardino de la Serna, J.; Barron, A. E. Biochim. Biophys. Acta, Biomembr. 2010, in press. (31) Kirshenbaum, K.; Barron, A. E.; Goldsmith, R. A.; Armand, P.; Bradley, E. K.; Truong, K. T. V.; Dill, K. A.; Cohen, F. E.; Zuckermann, R. N. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 4303– 4308. (32) Appella, D. H.; Christianson, L. A.; Karle, I. L.; Powell, D. R.; Gellman, S. H. J. Am. Chem. Soc. 1996, 118, 13071–13072. (33) Cheng, R. P.; Gellman, S. H.; DeGrado, W. F. Chem. ReV. 2001, 101, 3219–3232. (34) Hayen, A.; Schmitt, M. A.; Ngassa, F.; Thomasson, K. A.; Gellman, S. H. Angew. Chem., Int. Ed. 2004, 43, 505–510. (35) Horne, W. S.; Gellman, S. H. Acc. Chem. Res. 2008, 41, 1399–1408. (36) Zuckermann, R. N.; Kerr, J. M.; Kent, S. B. H.; Moos, W. H. J. Am. Chem. Soc. 1992, 114, 10646–10647. (37) Gelman, M. A.; Lynn, D. M.; Weisblum, B.; Gellman, S. H. Org. Lett. 2004, 6, 557–60. (38) Schmitt, M. A.; Weisblum, B.; Gellman, S. H. J. Am. Chem. Soc. 2007, 129, 417–428. (39) Shai, Y. Biochim. Biophys. Acta 1999, 1462, 55–70.

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Figure 1. Monomers (A) and polymers (B) examined in this work. The copolymers are sequence-random. Because the β-lactams are racemic, the polymers are heterochiral and stereorandom. The C-terminal imide unit is derived from the β-lactam(s) used in the polymerization reaction. Thus, for each copolymer, R′ and R′′ could correspond to either of the β-lactam precursors. All polymers are cationic and were isolated as trifluoroacetate salts. Nomenclature, characterization, and synthesis details are presented in the Supporting Information (Table S1).

of helical host-defense peptides.40-42 Based on this finding, we hypothesized that nylon-3 copolymers might be able to mimic lung surfactant proteins SP-B and SP-C, the function of which depends upon interaction with lipids,18 as is true of natural antimicrobial peptides. Here, we describe flexible, sequence-random nylon-3 copolymers that are intended to mimic lung surfactant protein B. The efficacy of the designs was estimated by in vitro surfactant behavior in a mixed lipid film. Surface activity has been evaluated by pulsating bubble surfactometry (PBS)43,44 of copolymers in a Tanaka lipid (TL) film45 (1,2-diacyl-sn-glycero3-phosphocholine (DPPC)/1-palmitoyl-2-oleoyl-sn-glycero-3[phospho-rac-(1-glycerol)] (POPG)/palmitic acid (PA) 68:22:9 by weight). Our materials, which were synthesized via anionic ring-opening polymerization of β-lactams,42,46 display increased surface-active behavior relative to known peptide- and peptoidbased SP-B mimics, with values approaching that of porcinederived SP-B in the TL film.30 These nylon-3 copolymers display lower toxicity toward mammalian cells than do an SP(40) Mowery, B. P.; Lee, S. E.; Kissounko, D. A.; Epand, R. F.; Epand, R. M.; Weisblum, B.; Stahl, S. S.; Gellman, S. H. J. Am. Chem. Soc. 2007, 129, 15474–15476. (41) Epand, R. F.; Mowery, B. P.; Lee, S. E.; Stahl, S. S.; Lehrer, R. I.; Gellman, S. H. J. Mol. Biol. 2008, 379, 38–50. (42) Mowery, B. P.; Lindner, A. H.; Weisblum, B.; Stahl, S. S.; Gellman, S. H. J. Am. Chem. Soc. 2009, 131, 9735–9745. (43) Putz, G.; Goerke, J.; Taeusch, H. W.; Clements, J. A. J. Appl. Physiol. 1994, 76, 1425–1431. (44) Seurynck, S. L.; Brown, N. J.; Wu, C. W.; Germino, K. W.; Kohlmeir, E. K.; Ingenito, E. P.; Glucksberg, M. R.; Barron, A. E.; Johnson, M. J. Appl. Physiol. 2005, 99, 624–633. (45) Tanaka, Y.; Takei, T.; Aiba, T.; Masuda, K.; Kiuchi, A.; Fujiwara, T. J. Lipid Res. 1986, 27, 475–485. (46) Zhang, J.; Kissounko, D. A.; Lee, S. E.; Gellman, S. H.; Stahl, S. S. J. Am. Chem. Soc. 2009, 131, 1589–1597.

B-derived peptide and a peptoid-based mimic. Our polymers are heterochiral because they were prepared from racemic β-lactams; therefore, these nylon-3 copolymers presumably cannot adopt specific, regular, or patterned conformations. Thus, our results challenge the notion that a helix or other regular conformation is strictly required if a molecule is to achieve global amphiphilicity (i.e., global segregation of lipophilic and hydrophilic subunits), a property that is thought to be necessary for SP-B-like activity. We postulate that the nylon-3 copolymers are able to achieve global amphiphilicity in irregular conformations after association with lipids, which facilitate their surfactant behavior. Results Copolymer Design, Synthesis, and Characterization. All monomers and polymers described here were prepared using previously reported procedures;40,42,46 their chemical structures are presented in Figure 1. The main variables in copolymer design were N-terminal modification and subunit composition. The β-lactams we employed gave rise both to lipophilic subunits, CH (for “cyclohexyl”) or CO (“cyclooctyl”) and to cationic subunits, MM (“monomethyl”) and DM (“dimethyl”). Incorporation of these subunits ensured that our polymers bear a net positive charge in aqueous solution and are amphiphilic, as is true of SP-B itself. However, in contrast to SP-B, our polymers presumably cannot achieve global segregation of lipophilic and cationic side chains by adopting a specific, regular, or patterned conformation because the sequence of lipophilic and cationic subunits varies among polymer molecules. In addition, because the β-lactam monomers are racemic, stereochemistry varies among polymer chains. The CH and CO subunits in our nylon-3 copolymers were intended to mimic the roles of lipophilic side chains and J. AM. CHEM. SOC.

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cyclically constrained residues in SP-B. The so-called “insertion region” of human SP-B, residues 1-9, contains a high proportion of residues that are aromatic and lipophilic (Phe1, Tyr5, and Trp8) or that are cyclically constrained (Pro2, Pro4, and Pro6). In previous work, this region was shown to be critical for the γ-reducing behavior of SP-B.47,48 Furthermore, when aromatic residues or prolines were substituted with alanine, the resulting peptides showed significantly decreased surface activity.48 It has been hypothesized that the N-terminal region of SP-B inserts transiently into the lipid layer with Trp as an anchor,49 thus allowing the protein to function overall as a lipid organizer and transporter. The designations we employed for the nylon-3 polymers (Figure 1) indicate the subunit identities and proportions, the latter determined by the ratio of β-lactams used in the polymerization reaction. The subunit sequence is random within the copolymers, and because the β-lactams were racemic, each polymer sample contains a mixture of backbone configurations. Most of the polymers have a p-(tert-butyl)benzoyl group at the N-terminus, but two of the copolymers were prepared with an octadecanoyl group at the N-terminus in an attempt to introduce an SP-C-like lipophilic tail into our SP-B mimetic polymers. We previously examined peptoid-based SP-C analogues that bear one or two octadecyl groups at the N-terminus.50 The favorable impact of the lipophilic tail(s) on surface activity prompted us to examine similar modifications of peptoid-based SP-B analogues, and again, we observed a marked improvement in surfactant activities relative to unalkylated analogues.30 These findings with sequence-specific surfactant protein mimics led us to explore analogous N-terminal modifications of nylon-3 copolymers. The β-lactams required for nylon-3 copolymer synthesis were prepared by previously published methods involving the [2 + 2] cycloaddition of chlorosulfonyl isothiocyanate (CSI) to alkenes. Anionic ring-opening copolymerization of the βlactams51,52 followed by acid-mediated removal of the tert-butyl carbamate protecting groups yielded nylon-3 materials that are cationic at neutral pH.40 Gel permeation chromatography (GPC) performed before deprotection indicated polydispersity indices (PDI) in the range 1.04-1.18 (see the Supporting Information). We were unable to measure polydispersity after side-chain deprotection, but we assume that this process does not alter polydispersity. NMR measurements suggested that deprotection reactions proceeded to completion (see the Supporting Information). Copolymers identified as promising in early PBS screenings (1:2 and 2:1 MM:CO and 1:1 and 2:1 DM:CO) were resynthesized to establish the reproducibility of the polymer preparation protocol, as manifested in surface activity and cytotoxicity. For these polymers, different batches are denoted as a, b, and c. Pulsating Bubble Surfactometry: Static-Bubble Mode. The immediate adsorptive, γ-reducing effect, and stability of the films over time at a bubble a/l interface were assessed via PBS in static-bubble mode. Polymers dried with lipids (Tanaka Lipids, TL, DPPC/POPG/PA 68:22:9 [wt/wt/wt]) were sus(47) Ryan, M. A.; Qi, X.; Serrano, A. G.; Ikegami, M.; Perez-Gil, J.; Johansson, J.; Weaver, T. E. Biochemistry 2005, 44, 861–872. (48) Serrano, A. G.; Ryan, M. A.; Weaver, T. E.; Perez-Gil, J. Biophys. J. 2006, 90, 238–249. (49) Wang, Y. D.; Rao, K. M. K.; Demchuk, E. Biochemistry 2003, 42, 4015–4027. (50) Brown, N. J.; Bernardino de la Serna, J.; Barron, A. E. Biophys. J. 2010, in press. (51) Hashimoto, K. Prog. Polym. Sci. 2000, 25, 1411–1462. (52) Cheng, J. J.; Deming, T. J. J. Am. Chem. Soc. 2001, 123, 9457–9458. 7960

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Figure 2. PBS adsorption plot for lipid-polymer films in static-bubble mode at 37 °C. Representative static-bubble adsorption traces for Tanaka lipids alone (TL) and TL + 10 rel wt % of each mimic in an aqueous buffer (150 mM NaCl, 10 mM HEPES, 5 mM CaCl2, pH 6.9) suspension at 37 °C.

pended in aqueous buffer (150 mM NaCl, 10 mM HEPES, 5 mM CaCl2, pH 6.9) at 37 °C and allowed to adsorb to the interface of a 0.4 mm radius bubble for 20 min, yielding γ as a function of time. In this study, it is crucial for surface-active films to both adsorb rapidly and reach a low “equilibrium” or final γ in the time tested. Therefore, a mimic is considered very surface-active in the lipid film if it adsorbs to ∼25 mN m-1 within ∼1-5 min; for instance, Infasurf, an animal-derived SRT, attains a low γ ∼ 23 mN m-1 within ∼1-2 min on the PBS,44 and TL + porcine-derived SP-B films reach ∼26 mN m-1 at 5 min.30 Select polymer adsorption traces are depicted in Figure 2, while the adsorbed γ of all the lipid films containing polymers and positive controls, including lipids alone (TL), the peptides SP-B1-25, and KL4 (the latter is a peptide-based SP-B mimic with broad, biomimetic cationic residue patterning),53 and aromatic-rich peptoid B1,20 are presented at 5 min adsorption in Figure 3, panels A-D. Mean adsorptions (γ) ( standard deviation (σ) at selected time intervals for all films are available in the Supporting Information (Tables S2 and S3). For staticbubble experiments, relative closeness of γ values for different lipid-polymer films was largely determined by comparing the σ of the mean γ for the films; however, as a guideline, we consider that a difference of g2 mN m-1 among adsorptive γ values of different films is significant. Any additive to the lipid film significantly improved adsorptive characteristics relative to TL alone, with TL + KL4 exhibiting the most positive surfactant activity among the positive controls, reaching 22 mN m-1 at 5 min (Figure 3, panel A). Control TL + SP-B1-25 was more active than TL + B1, but both were less active than TL + KL4 (see adsorptive traces for positive control films in Supporting Information, Figure S29). All of the nylon-3 polymers displayed surface activity in this assay. Among the nylon-3 homopolymers, TL + DM yielded a lower γ than TL + MM, and an analogous trend was evident among the films containing MM:DM copolymers. The introduction of lipophilic CH subunits to polymers in the lipid films moderately improved the γ values reached (Figure 3, panel B, and Supporting Information, Table S2) but impeded the adsorption rate relative to films containing polymers with entirely (53) Cochrane, C. G.; Revak, S. D. Science 1991, 254, 566–568.

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Figure 3. PBS adsorption data for lipid-polymer films in static-bubble mode at 37 °C. Mean γ (mN m-1) at 5 min adsorption are presented for all films including polymers or positive controls in panels A-D. Error bars are the standard deviation of the mean (σ). See the Supporting Information (Tables S2 and S3) for mean adsorption γ data ( σ at selected time intervals.

cationic subunits (MM homopolymer, DM homopolymer, or MM:DM copolymers). Among CH-containing copolymer-lipid films, an increasing proportion of lipophilic CH subunits (1:2 MM:CH vs 2:1 MM:CH) or replacement of MM subunits with slightly more lipophilic DM subunits (2:1 DM:CH vs 2:1 MM: CH) also resulted in lower γ values during adsorption (Figure 3, panel B, and Supporting Information, Table S2). The best surface activities were observed among films with nylon-3 copolymers containing CO subunits. Both TL + 1:1 DM:CO and TL + 1:2 DM:CO displayed excellent activities, with γ reaching 26-28 mN m-1 after as little as 2.5 min of adsorption (Figure 2; Supporting Information, Table S2).

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Replacing the p-(tert-butyl)benzoyl group at the N-terminus with an octadecanoyl group did not affect PBS static-mode surface activity (films containing 1:1 DM:CO copolymers). Raising the cationic subunit proportion to 67% led to a significant decrease in surface activity, and among these polymers, the use of the DM subunit provided improvement relative to use of the MM subunit (2:1 DM:CO vs 2:1 MM:CO). In addition, the adsorptive activities of the nylon-3 copolymers were reproduced in subsequent batches (Figure 3, panels A-D; also see the Supporting Information, Tables S2 and S3). Pulsating Bubble Surfactometry: Dynamic-Bubble Mode. The dynamic surfactant activity during changes in volume or film surface area provides an indication of film sustainability over time. Bubble pulsation at the approximate adult respiratory rate of 20 cycles per minute (cpm) in PBS dynamic-bubble mode at 37 °C permits a simplified evaluation of such dynamic film behavior.44 In dynamic mode, after static-bubble adsorption, the bubble was subsequently pulsed for 10 min and γ was recorded with respect to surface area. In Figure 4, panels A-D depict the attained maximum and minimum γ (γmax/min) at 5 min of cycling for all lipid films containing polymers or positive controls. Mean γmax/min values ( σ at selected time intervals for up to 10 min of cycling are located in the Supporting Information (Tables S4 and S5). Representative single pulsation γ-surface area (SA) bubble hysteresis loops at 5 min cycling for select lipid-polymer films are presented in Figure 6, panels A-D. Bubble expansion corresponds to a clockwise loop direction and vice versa for compression. The absence of low-γ data in some loops is caused by the limited ability of the image analysis system to trace the bubble shape in this regime.44 The highly compressed state of the film, which enables it to reach near-zero γ, often causes significant bubble shape deformation. This deformation may prevent the level of bubble tracing needed to obtain SA and to calculate γ via the ellipsoidal Laplace equation.44 However, visual, real-time bubble inspection during the experiment confirmed that γ reached near-zero in these films.44 In addition, lipid-polymer films that did not reach